We have identified a frequent mutation at nucleotide 908 of ER1 (A908G) in over 30% of women with hyperplasia of the breast, and strikingly in 50% of invasive breast cancers. This mutation resulted in an amino acid substitution of arginine for lysine at residue 303 (K303R ER1), which conferred the ability for enhanced breast tumor cell growth in low levels of estrogen. Upon estrogen withdrawal, mutant-expressing tumors regress, but eventually regrow, suggesting that the mutation also confers resistance to estrogen ablation therapies. When wild type (WT) ER1-overexpressing breast cancer cells were stimulated with growth factors, cells became resistant to the growth-inhibitory effects of the antiestrogen tamoxifen (Tam). In contrast, growth factor stimulation of K303R ER1-expressing cells resulted in Tam acting as a growth agonist. The agonist hypersensitive phenotype of the mutant was associated with decreased protein acetylation, increased receptor coactivator binding, decreased corepressor binding, and increased promoter occupancy of estrogen-regulated genes. The mutation also generated an enhanced phosphorylation substrate for the intracellular signaling kinase pathways AKT, PAK-1, and PKA. Recently, we have discovered that the mutation confers resistance to an aromatase inhibitor as well. Our collective data suggest that the hinge domain where the mutation resides plays a pivotal and potentially interdependent role between other functional domains of the ER1. We therefore hypothesize that the mutation adapts ER1 for enhanced reception of intracellular signal transduction, which confers resistance to hormonal therapies. Our proposed Aims are: (1) To determine the distinct biological consequences of specific ER1 phosphorylation events in the evolution of hormone resistance. We will generate mutations at serines 118 and 305, and tyrosine 537 in ER1. Altered function and localization will be assessed. We will generate aromatase and K303R co-expressing models to be used in anchorage independent assays and xenograft models. We will cross K303R ER1 transgenic mice with dominant active AKT and PKA mice to assess effects of phosphorylation on ER function. (2) To examine the role of the K303R ER1 mutation on the emergence of resistance to estrogen deprivation. We will perform expression microarray analyses, and luciferase reporter assays to determine the effects of phosphorylation. Aromatase co-expressing lines have been developed and will be used in xenograft tumor studies. (3) To determine the impact of phosphorylation and mutation on the luminal breast cancer phenotype and the problem of hormone resistance. We compare the frequency of the K303R ER1 mutant in microarray profiles to determine if the mutant comprises the poor prognosis, luminal B subtype. We will examine the mutation in a retrospective predictive study of patients using sequence analysis and phosphorylation-specific ER antibodies. PUBLIC HEALTH RELEVANCE: We discovered a mutation in the estrogen receptor genes which makes it very sensitive to the low levels of hormone, like the levels found in postmenopausal women. We will test whether the mutation causes a women to not respond to common therapies, such as tamoxifen and an aromatase inhibitor. Our goal is to help women decide which therapy is best for them, and to reduce the risk of relapse and recurrence of breast cancer.